Proline is a non-essential amino acid and Sahu et al. show that some cancer cells are dependent on proline for clonogenicity and tumorigenic potential. These authors profiled a panel of cancer cell lines and found that proline consumption and the expression of enzymes involved in proline biosynthesis correlated with clonogenicity and tumorigenic potential. Those cancer cell lines with a dependency on proline had hyperactivation of the mTOR complex 1 (mTORC1)–4EBP1 pathway and endoplasmic reticulum stress. These data indicate that targeting proline biosynthesis and uptake may be effective in some types of cancer.